The goal of this competitive renewal application is to increase the clinical utility of labeled mAbs for cancer diagnosis and therapy by developing more effective strategies for labeling internalizing mAbs and mAb fragments with radioiodine nuclides and 211At. With the emergence of the epidermal growth factor receptor variant III (EGFRvIII) as a tumor-specific molecular target on glioma and other tumors, research during the previous grant period was focused on the development of strategies for labeling mAbs reactive with this rapidly internalizing mutant receptor. Our studies revealed that radio iodination of anti-EGFRvIII mAbs using reagents containing benzoates bearing charged substituent's or D-amino acid peptides significantly improved retention of radioactivity in EGFRvIII-expressing tumor cells and xenografts compared with mAbs labeled by conventional approaches. The most promising results were obtained with N5-(3-[*I]iodobenzoyl)-Lys5-N1-maleimido-D-Gly1- GEEEK ([*I]IB-Mal-D-GEEEK), which includes 3 D-glutamates to provide a negatively charged, proteolytically inert moiety and an iodobenzoyl group to minimize dehalogenation. Our hypothesis is that optimized labeling methods for internalizing mAbs such as anti-EGFRvIII based on the IB-Mal-D-GEEEK template will enhance tumor retention and tumor-to-normal tissue ratios, thereby improving their clinical potential as diagnostic and therapeutic agents. We propose to: 1) label anti-EGFRvIII mAbs and fragments with radioiodine nuclides and 211At using IB-Mal-D-GEEEK and its [211At]astatobenzoyl Mal-D-GEEEK analogue and to evaluate their potential as diagnostic and therapeutic radiopharmaceuticals; 2) investigate strategies for improving the Mal- GEEEK reagent for labeling internalizing mAbs including alteration in D-peptide sequence (number and nature of negatively charged amino acids), use of a less hydrophobic dehalogenation resistant prosthetic group such as iodopyridine, incorporation of a cathepsin B cleavable linker, and coupling the radiohalogenation precursor to the mAb prior to the labeling reaction; 3) investigate the nature of the low and high molecular weight labeled catabolites generated in tumor cells in vitro and tumor and normal tissues in vivo; 4) evaluate the therapeutic efficacy of promising 131I- and 211At-labeled anti-EGFRvIII conjugates in athymic rodents with subcutaneous, intracranial, and neoplastic meningitis xenografts; and 5) With the best radio labeled anti-EGFRvIII conjugates, conduct all the toxicity, efficacy, dosimetry, and other FDA-required studies for Investigational New Drug permits, to all performance of clinical trials in malignant glioma patients under our Brain Tumor Center grant (NS20023